There is a clear need for alternative chemotherapeutic strategies for Toxoplasma gondii and related apicomplexan parasites. Invasion by these parasites is morphologically similar throughout the phylum and is accompanied by proteolytic processing of the contents of the microneme organelle. Serine proteinase inhibitors have been recently shown to block host cell invasion by T. gondii. Proteinase inhibitors have been highly successful in a variety of pathogenic conditions. T. gondii will be used as a model system to evaluate the suitability of subtilisin-like serine proteinases (subtilases) as chemotherapeutic targets in the Apicomplexan parasites. Two subtilases from T. gondii have been cloned and at least one of these may localize to the micronemes. Preliminary studies indicate that the serine proteinase inhibitor DFP blocks processing of microneme proteins H4 and MIC2. It is hypothesized that T. gondii subtilase(s) mediate invasion of host cells and that inhibition of subtilases will prove useful for treatment of infections with other important Apicomplexan parasites. This proposal will complete the cloning and characterization of subtilase genes from Toxoplasma gondii. The pattern of synthesis, processing and subcellular localization of the subtilases will be clarified using antibody to proteinases and epitope-tagged proteins. Enzymatically active recombinant subtilases will be expressed. Tachyzoite cell fractions and recombinant proteinases will be tested for proteolytic cleavage of artificial substrates. These substrates will be used to characterize the inhibitor profile and substrate specificity of T. gondii subtilases. The function of the subtilases will be determined using gene disruption. The ability of knockout parasites to invade host cells, process microneme proteins, and cause disease in animals will be tested. Finally, it will be determined if the microneme proteins H4 and MIC2 are proteolytically cleaved by T. gondii subtilases prior to or during invasion. These studies will further understanding of invasion by Apicomplexan parasites and will lead to novel chemotherapeutic strategies for these organisms.